RU2697096C1 - Method for identification of genetic polymorphisms affecting metabolism of anticancer drugs using biological microchips - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used in medicine. Invention refers to a method for detection of nucleotide replacements in genes influencing metabolism of chemotherapeutic preparations. Invention includes a biological microchip (biochip), a set of oligonucleotide probes and primers for implementing the method. Identification of polymorphisms is carried out by amplifying gene fragments with polymorphic loci using a set of primers and subsequent hybridization of amplification products on a biochip with immobilized oligonucleotide probes.

EFFECT: disclosed is a method for identifying genetic polymorphisms affecting the metabolism of antineoplastic preparations using biological microchips.

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Description

FIELD OF THE INVENTION

The invention relates to the field of molecular biology, genetics and pharmacogenomics and provides a method for identifying genetic polymorphisms that affect the metabolism of antitumor drugs using biological microarrays.

State of the art

Currently, there is a large arsenal of methods for analyzing genetic polymorphisms. Conventionally, the following approaches can be distinguished:

1) Sequencing of target fragments carrying a polymorphic region. Sanger sequencing is the reference and most accurate method for detecting genetic polymorphisms. The method consists in carrying out a polymerase chain reaction (PCR) in the presence of fluorescently-labeled dideoxynucleoside triphosphates (ddNTP) and the subsequent separation of the fragments synthesized during the reaction by electrophoresis with detection of the fluorescent signal at the output. The advantage of this method is its high accuracy and sensitivity, the main disadvantage is the high cost of equipment (Yuan ZJ, Zhou WW, Liu W., et al. Association of GSTP1 and RRM1 polymorphisms with the response and toxicity of gemcitabine-cisplatin combination chemotherapy in Chinese patients with non-small cell lung cancer. Asian Race J. Cancer. Prev. 2015, v. 16 (10), p. 4347-4351; Zhou F., Yu Z., Jiang T., et al. Genetic polymorphisms of GSTP1 and XRCC1: prediction of clinical outcome of platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients. Swiss Med Wkly. 2011 v. 141, p. 13275-13284).

2) Methods using a primer extension strategy. Among these methods, methods using a common primer for detecting both alleles and methods using specific primers for each of the alleles are distinguished.

In the methods of elongation of the common primer (common primer extension, CPE), the primer is selected so that its 3'-end is annealed in position immediately in front of the site of single nucleotide polymorphism (SNP). During the reaction, the primer is completed with a nucleotide complementary to the corresponding allelic variant of SNP (Sokolov B.P. Primer extension technique for the detection of single nucleotide in genomic DNA. Nucleic. Acids. Res. 1990, v. 18 (12), p. 3671) . The inserted nucleotide is determined by MALDI-TOF mass spectrometry (Ross P., Hall L., Smirnov I., Haff L. High level multiplex genotyping by MALDI-TOF mass spectrometry. Nat. Biotechnol. 1998, v. 16 (13), p . 1347-1351; Haff LA, Smirnov LP. Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry. Genome. Res. 1997, v. 7 (4), p. 378-388) . As embedded nucleotides, ddNTP is used, which leads to the elongation of the primer by one nucleotide (single base extension, SBE). After the reaction, the products are detected using MALDI-TOF mass spectrometry. The disadvantage of the SBE method is the low resolution in the analysis of A / T heterozygous genotypes. An improved version of the SBE method using modified ddNTPs is presented in (Fei Z., Opo T., Smith LM MALDI-TOF mass spectrometric typing of single nucleotide polymorphisms with mass-tagged ddNTPs. Nucleic. Acids. Res. 1998, v. 26 ( 11), p. 2827-2828). In the MassEXTEND method (Sequenom, USA), a mixture of dNTP and ddNTP is used to extend the primers. This approach increases the mass difference between the products corresponding to different allelic variants, which increases the accuracy of the method. A high-performance automated version of this method is implemented on the MassARRAY platform (Sequenom, USA) (Yang Z., Fang X., Pei X., Li H. Polymorphisms in the ERCC1 and XPF genes and risk of breast cancer in a Chinese population. Genet. Test Mol. Biomarkers. 2013, v. 17 (9), p. 700-706). In the method presented in (Berlin K., Gut IG Analysis of negatively 'charge tagged' DNA by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. Rapid. Commun. Mass. Spectrom. 1999, v. 13 (17), p. 1739-1743), primers modified at the 3'-end of the phosphotionate are used, and aS-ddNTP is used as nucleotides for elongation. As a result of the reaction, DNA-labeled fragments are generated. Thanks to this approach, preliminary purification of the reaction products before mass spectrometry is not required, and the resolution of the analysis is also increased. A common drawback of MALDI-TOF mass spectrometry detection methods is the use of expensive equipment and the need for highly qualified personnel, which limits their use in clinical practice.

In methods for elongation of a common primer with detection by fluorescence, fluorescently-labeled dTNTs are used as termination nucleotides. These techniques are implemented in several formats: homogeneous reaction and detection; solid phase detection; solid phase reaction. SNaPshot (Applied Biosystems, USA) uses standard PCR with primer elongation on one fluorescently-labeled dTNP, the products of which are then separated and detected by capillary electrophoresis (Le Hellard S., Ballereau SJ, Visscher PM, et al. SNP genotyping on poo : comparison of genotyping technologies and a semi-automated method for data storage and analysis. Nucleic. Acids. Res. 2002, v. 30 (15), p. 74-84; Ryu JS, Hong YC, Han HS, et al. Association between polymorphisms of ERCC1 and XPD and survival in non-small-cell lung cancer patients treated with cisplatin combination chemotherapy. Lung. Cancer. 2004, v. 44 (3), p. 311-316). (Fan JB, Chen X., Halushka MK, et al. Parallel genotyping of human SNPs using generic high density oligonucleotide tag arrays. Genome. Res. 2000, v. 10 (6), p. 853-860) used primers with adapters in a homogeneous reaction, and the products of primer elongation hybridize with oligonucleotides immobilized on a solid support, complementary to the adapters. Genotype determination is carried out by detecting a fluorescent signal on a substrate. The solid phase reaction is used in the method of elongation of oligonucleotides attached to a substrate (arrayed primer extension, APEX). PCR products of the target gene fragments hybridize with oligonucleotides and complete with fluorescently-labeled dTNPs (Pullat J., Metspalu A. Arrayed primer extension reaction for genotyping on oligonucleotide microarray. Methods. Mol. Biol. 2008, v. 444, p. 161-167) . In (Shapero MH, Leuther KK, Nguyen A., et al. SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing. Genome. Res. 2001, v. 11 (11), p. 1926-1934) PCR and single nucleotide primer extension is carried out directly on the substrate using the same set of primers. The advantage of reactions on the solid phase is the absence of inter-primer interactions, which increases the sensitivity and specificity of the analysis. The main disadvantages of approaches with detection or reaction on the solid phase include the high cost of consumables and equipment. The disadvantage of the methods performed in the liquid phase is the inability to simultaneously identify multiple targets.

In methods using specific primers for each of the alleles, primers are used that differ only in the last nucleotide at the 3'-end. Their elongation occurs if their 3'-terminal nucleotide is complementary to the nucleotide at the SNP site, which allows discriminating allelic variants of polymorphisms. This group of methods includes various modifications of allele-specific PCR (Allele-specific PCR, AS-PCR). The combination of allele-specific PCR with real-time PCR followed by separation of products by capillary electrophoresis is presented in (Medintz I., Wong WW, Berti L., et al. High-performance multiplex SNP analysis of three hemochromatosis-related mutations with capillary array electrophoresis microplates. Genome. Res. 2001, v. 11 (3), p. 413-421). In the method of elongation of allele-specific primers (Allele-specific primer extension, ASPE), pre-amplified target fragments are used, on which the primer is elongated with the inclusion of a fluorescent label introduced into the reaction mixture (Ugozzoli L., Wahlqvist JM, Ehsani A., et al al Detection of specific alleles by using allele-specific primer extension followed by capture on solid support. Genet. Anal. Tech. Appl. 1992, v. 9 (4), p. 107-112). In (Bortolin S., Black M., Modi H., et al. Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single- nucleotide polymorphisms. Clin. Chem. 2004, v. 50 (11), p. 2028-2036) primers are used for elongation with adapters that are terminated with fluorescently-labeled dNTPs. After hybridization of the reaction products with oligonucleotides immobilized on microspheres and complementary to primer adapters, their flow cytometry is performed. The disadvantage of methods based on allele-specific PCR is the limitation on the number of simultaneously determined polymorphisms - as a rule, in such methods no more than 3-5 targets are determined, and a reduced, close to subjective, resolution when interpreting the results.

3) Methods based on hybridization. These methods use a difference in the melting temperatures of perfect and imperfect duplexes between the target carrying the SNP site and the detecting oligonucleotides (probes), which makes it possible to identify allelic variants of polymorphisms. Discrimination of allelic variants in hybridization-based methods depends on various factors, including: length and sequence of the probe; the location of the SNP site in the probe; hybridization conditions.

Hybridization-based methods are used in high-performance microchip-based platforms. GeneChip microarray technology (Affymetrix, USA) uses allele-specific probes with a length of 25 bases, which are synthesized directly on a substrate by photolithography. Target gene fragments carrying SNPs are amplified by PCR, cleaved into shorter fragments, labeled with biotin tags, and hybridized on a microchip. Visualization of the results of hybridization is carried out using fluorescence labeling with dye with streptavidin. Several millions of different oligonucleotide probes can be located on each microchip, which allows the determination of 10 ⁴ -10 ⁵ SNPs in a single analysis (Matsuzaki N., Dong S., Loi N., et al. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods. 2004, v. 1 (2), p. 109-111). The disadvantage of this method is the high cost of consumables and the complexity of the interpretation of the obtained fluorescence images, which dramatically limits the application of this method in clinical practice.

An alternative approach using oligonucleotide microarrays is represented by the use of low density microarrays. Methods using them differ in the method of deposition of oligonucleotides, the type of substrate and the procedure for detecting hybridization results. One of the methods belonging to this group is a two-stage multiplex PCR followed by hybridization of the reaction products on low-density hydrogel microchips. At the first stage, PCR is carried out with equal concentrations of forward and reverse primers to generate fragments of genes carrying the analyzed SNPs. In the second stage, the product of the first stage is used as a template for PCR, but fluorescence-labeled dNTP is added to the reaction mixture. It should be noted that in order to produce a single-stranded DNA fragment necessary for hybridization, an unequal concentration of forward and reverse primers (asymmetric PCR) is used in the reaction. Hybridization of a single-stranded DNA fragment is carried out on hydrogel microarrays. Hydrogel microchips are plastic substrates with immobilized hydrogel cells. Specific oligonucleotide probes are covalently fixed in each cell with which products of the second stage of PCR hybridize (Heydarov R., Titov S., Abramov M., Timofeev E., Mikhailovich V. Hydrogel microarray for detection of polymorphisms in the UGT1A1, DP YD, GSTP1 and ABCB1 genes. Cancer. Biomark. 2017, v. 18 (3), p. 265-272). A modification of this method is the use of locked nucleic acid (LNA) oligonucleotides as probes. Duplexes of such oligonucleotides have increased thermal stability and provide significantly higher discrimination of hybridization signals (Fesenko E.E., Heydarov RN, Stepanova EV, et al. Microarray with LNA probes for genotyping of polymorphic variants of Gilbert's syndrome gene UGT1 Al (TA) n Clin. Chem. Lab. Med. 2013, v. 51 (6), p. 1177-1184). The presented method is the closest analogue, inferior in diagnostic characteristics, and a prototype of the method proposed in this invention. The advantages of the above method include: the low price of consumables, the relative ease of analysis and interpretation of the results. The main disadvantage is the small (compared with the proposed method) the number of simultaneously identified SNPs.

One of the widely used methods for determining SNPs is TaqMan technology (Applied Biosystems, USA), which combines hybridization, the use of 5'-exonuclease activity of polymerase, followed by fluorescence detection. Four oligonucleotides are used in this method: two allele-specific probes that differ in position corresponding to the SNP site, and two PCR primers flanking the SNP site. Allele-specific probes carry a fluorescent label at one end and a fluorescence quencher at the other end. In a non-responsive loop-coiled state, such probes do not fluoresce due to the proximity of the tag and the quencher. For the detection of SNPs by TaqMan, a polymerase with 5'-exonuclease activity is used. In the process of elongation of primers, the polymerase cleaves the fluorescently-labeled nucleotide only from the probe, which is completely complementary to the sequence of the target site, which leads to the accumulation of the fluorescent signal due to its release into the solution. The accumulation of the fluorescent signal determines the allelic variant of the analyzed target (Ludovini V., Floriani I., Pistola L., et al. Association of cytidine deaminase and xeroderma pigmentosum group D polymorphisms with response, toxicity, and survival in cisplatin / gemcitabine-treated advanced non non -small cell lung cancer patients. J. Thorac. Oncol. 2011, v. 6 (12), p. 2018-2026; Lv H., Han T., Shi X., et al. Genetic polymorphism of GSTP1 and ERCC1 correlated with response to platinum-based chemotherapy in non-small cell lung cancer. Med. Oncol. 2014, v. 31 (8), p. 86-93). The disadvantages of this method include the low scalability and the need for expensive equipment for analysis.

Another method belonging to this group is dynamic allele-specific hybridization. The method consists in analyzing the rate of denaturation of the probe and target duplexes in real time by fixing the level of fluorescence. At the preparatory stage, the target fragment carrying the SNP is amplified with primers, one of which is modified with biotin, which allows the single-chain PCR product to be coupled to streptavidin-coated microspheres. After this, hybridization with allele-specific probes is carried out in the presence of an intercalating fluorescent dye. Further, the temperature rises and the rate of denaturation of duplexes is fixed. The melting curve has a different shape for perfect and imperfect duplexes, and the allelic variant of the analyzed SNP is determined from the shape of this curve (Howell WM, Jobs M., Gyllensten U., Brookes A J. Dynamic allele-specific hybridization. A new method for scoring single nucleotide polymorphisms. Nat. Biotechnol. 1999, v. 17 (1), p. 87-88). The disadvantages of the method include its relative complexity and complexity with the simultaneous determination of several SNPs.

4) Ligase detection reaction (LDR) based methods. These methods employ highly specific ligase enzymes that crosslink a single stranded gap between adjacent oligonucleotide probes linked to the target fragment at the SNP site. The reaction uses 3 probes, two of which are allele-specific, and the third probe is common. The ligase reaction products are subsequently detected by electrophoresis. Most methods based on the ligase reaction use allele-specific oligonucleotide probes of the 3'-terminal nucleotide which are directly complementary to the SNP site, since ligases are most sensitive to unpaired nucleotides at the 3'-end (von Keyserling N., Bergmann T., Schuetz M., et al. Analysis of 4 single-nucleotide polymorphisms in relation to cervical dysplasia and cancer development using a high-throughput ligation-detection reaction procedure. Int. J. Gynecol. Cancer. 2011, v. 21 (9), p . 1664-1671.). Despite the high specificity of this method (which can be attributed to its advantages), its significant drawback is the difficulty in selecting probes with the simultaneous detection of several SNPs. 5) Methods based on enzymatic cleavage. These methods are based on the ability of certain enzymes to break down DNA when recognizing specific sequences and secondary structures.

This group of methods includes analysis of restriction fragment length polymorphism (RFLP). The restriction endonucleases used in this method are selected so that they are specific to the SNP site. After preliminary amplification of the target sites and their purification, the PCR product is cleaved and the fragments obtained are separated by gel electrophoresis or blot hybridization. The advantages of this method include the simplicity of analysis and interpretation of the results, but a significant drawback is the limited choice of targets for genotyping, due to the need for specific restriction enzymes specific to the SNP site sequence (Liu JY, Liu QM, Li LR Association of GSTP1 and XRCC1 gene polymorphisms with clinical outcomes of patients with advanced non-small cell lung cancer. Genet. Mol. Res. 2015, v. 14 (3), p. 10331-10337; Shi ZH, Shi GY, Liu LG Polymorphisms in ERCC1 and XPF gene and response to chemotherapy and overall survival of non-small cell lung cancer. Int. J. Clin. Exp. Pathol. 2015, v. 8 (3), p. 3132-3137).

Another example of these methods is the invasive cleavage of an oligonucleotide probe (Invader assay). In this approach, a special Flap-endonuclease is used, which is able to recognize sections of DNA that come on one another. Three oligonucleotide probes participate in the reaction, two are allele-specific detecting probes, and the third is an invasive probe. The invasive probe is complementary to the 3'-site of the SNP site, at the end of this probe is not a complementary SNP nucleotide, but allele-specific probes to the 5'-site and carry a signal fragment at its 5'-end. Removal of the signal fragment by Flap-endonuclease occurs only in the case when a perfect duplex is formed between the site with SNP and a specific part of the detecting probe. Detection of the analysis results is carried out by recording the fluorescence of the cleaved signal fragment. A common drawback of enzymatic digestion methods is the inability to accurately identify SNPs (Olivier M. The Invader assay for SNP genotyping. Mutat. Res. 2005, v. 573 (1-2), p. 103-110).

6) Methods based on the physical properties of DNA molecules.

Conformational analysis of single-stranded DNA fragments (Single-strand conformation polymorphism, SSCP). Analysis by this method involves the preliminary amplification of polymorphic regions with their subsequent denaturation and separation of single-stranded fragments by gel electrophoresis. The mobility of single-stranded fragments during electrophoresis depends on their nucleotide composition, which determines the secondary and tertiary structure, which makes it possible to identify allelic variants of polymorphisms. The advantage of this method consists in the simplicity of the analysis and low cost, the main disadvantage is the low reproducibility of the results, poor scalability and the inability to uniquely identify the type of SNP (Akhtar S., Mahjabeen I., Akram Z., Kayani MA CYP1A1 and GSTP1 gene variations in breast cancer : a systematic review and case-control study. Fam. Cancer. 2016, v. 15 (2), p. 201-214).

In addition to the literature, there are a number of patents and patent applications describing various methods for detecting polymorphisms in one or more genes from the list: GSTP1 (polymorphism rs 1695), ABCB1 (polymorphism rs 1045642), MTHFR (polymorphism rs 1801133), FCGR3A (polymorphism rs 396991 ), SOD2 (polymorphism rs 4880), TP53 (polymorphism rs 1042522), ERCC1 (polymorphisms rs 11615, rs 3212986), XPC (polymorphism rs 2228001), EGF (polymorphism rs 4444903), UGT1A1 (polymorphism rs 8175D7, DP rs 3918290, rs 2297595, rs 67376798), XRCC1 (polymorphism rs 25487), CYP2D6 (polymorphism rs 3892097), TPMT (polymorphisms rs 1142345, rs 1800462, rs 1800584, rs 1800460).

Patents and patent applications describing methods for detecting polymorphisms in a single gene:

- the method described in patent application CN 104988226 A (from 2015-10-21), provides for the determination of polymorphism rs 1695 in the GSTP1 gene using the SNaPshot method;

- the method described in patent application CN 106755352 A (from 2017-05-31), provides for the determination of polymorphism rs 1045642 in the ABCB1 gene using allele-specific real-time PCR with fluorescence detection of the product;

- the method described in patent application CN 102851365 A (from 2012-08-31), provides for the determination of polymorphism rs 1801133 MTHFR using the allele-specific PCR method with the detection of the product by agarose gel electrophoresis;

- the method described in patent application CN 107227363 A (from 2017-10-03), provides for the determination of rs 11615 polymorphism in the ERCC1 gene using allele-specific PCR, peptide-nuclein oligonucleotides are used as specific primers;

- the method described in patent application CN 108103160 A (from 2018-06-01), provides for the determination of polymorphism rs 2228001 in the CSR gene using mass spectrometry of amplification products;

- the method described in patent US 6472157 B2 (from 2002-10-29), provides for the determination of polymorphism rs 8175347 in the UGT1A1 gene using PCR with subsequent separation of the reaction products by electrophoresis in polyacrylamide gel;

- the method described in patent application CN 107058608 A (from 2017-08-18), provides for the determination of polymorphism rs 3918290 in the DPYD gene using allele-specific real-time PCR with fluorescence detection of the product;

- the method described in patent application CN 104988225 A (from 2015-10-21), provides for the determination of polymorphism rs 25487 XRCC1 using the SNaPshot method;

- the method described in patent application CN 103131776 A (from 2013-06-05), provides for the determination of polymorphism rs 1142345 in the TPMT gene by PCR followed by sequencing of the reaction products;

- the method described in patent application CN 105506097 A (from 2016-04-20), provides for the determination of polymorphism rs 1142345 in the TPMT gene rs 1800462 using the SNaPshot method.

An obvious significant drawback of the above methods is the ability to detect only one SNP. The methods claimed in CN 108103160 A and CN 103131776 A require the use of expensive equipment.

Patents and patent applications describing methods for the simultaneous detection of multiple polymorphisms in different genes:

- the method described in patent application CN 104988224 A (from 2015-10-21), provides the detection of polymorphisms in the genes XRCC1 (rs 25487), ERCC1 (rs 11615) and GSTP1 (rs 1695) using the SNaPshot method.

- the method described in patent application CN 107663537 A (from 2018-02-06) allows detecting SNPs in 10 genes, including GSTP1 (rs 1695), MTHFR (rs 1801133), XRCC1 (rs 25487), ERCC1 (rs 11615), DPYD (rs 3918290) by solid-state multiplex PCR, followed by sequencing of individual amplicons;

- the method described in patent application CN 101275160 A (from 2008-10-01) allows identification of 48 SNPs in 45 genes, including ERCC1 (rs 11615), MTHFR (rs 1801133), XRCC1 (rs 25487 ), GSTP1 (rs 1695) by real-time PCR using TaqMan probes;

The main disadvantage of these methods is the high cost of equipment and the difficulty of interpreting the results of the analysis.

Disclosure of the invention

The present invention provides a method for identifying genetic polymorphisms in genes whose products affect the metabolism of antitumor drugs used in chemotherapy of malignant neoplasms using biological microarrays, which are a substrate with gel elements containing immobilized specific oligonucleotide probes. The invention provides: a biochip used in the method, a set of oligonucleotide probes used to make a biochip, as well as primers necessary for conducting a multiplex polymerase chain reaction. The method compares favorably with the methods known from the prior art in that it allows simultaneous identification of 20 clinically significant polymorphisms in 14 genes with high specificity. The method is devoid of the disadvantages indicated above when considering each method, does not require expensive equipment and materials.

The analysis of polymorphisms in the genes involved in the metabolism of antitumor drugs is important for assessing the risks of toxic effects of chemotherapy, selecting an individual treatment regimen and in predicting its outcome. The method can be used before prescribing a course of chemotherapy to increase the effectiveness of treatment of cancer patients, to correct treatment protocols, as a tool for analyzing the population in order to develop new protocols and recommendations for their use.

The procedure for determining polymorphisms in the proposed method is based on conducting multiplex polymerase chain reaction (PCR) using a DNA isolated from the patient’s peripheral blood as a matrix, followed by hybridization of the obtained single-stranded fluorescently-labeled PCR product on a biological microchip. A biological microchip is a substrate with orderly arranged hydrogel elements containing covalently immobilized oligonucleotide probes that hybridize to specific regions of genes carrying polymorphic nucleotide positions.

Hybridization results are detected by the fluorescence method by excitation of fluorescent labels embedded in the PCR product after its hybridization with specific probes immobilized on a microchip. Interpretation of the results is carried out by a comparative analysis of fluorescent signals in cells in which the labeled target is hybridized with immobilized probes.

The proposed method includes: a) providing a biochip, which is a substrate with ordered gel elements in which oligonucleotide probes are immobilized, belonging to the following groups:

- a group of probes, the sequences of which are presented by SEQ ID NO 1-4 (Table 1), to identify polymorphism rs 1695 (alleles A and G) in the GSTP1 gene;

- a group of probes, the sequences of which are presented by SEQ ID NO 5, 6 (Table 1), to identify polymorphism rs 1045642 (alleles T and C) in the ABCB1 gene;

- a group of probes, the sequences of which are represented by SEQ ID NO 7, 8 (Table 1), to identify polymorphism rs 1801133 (T and C alleles) in the MTHFR gene;

- a group of probes, the sequences of which are represented by SEQ ID NO 9, 10 (Table 1), to identify polymorphism rs 396991 (alleles G and T) in the FCGR3A gene;

- a group of probes, the sequences of which are presented by SEQ ID NO 11-14 (Table 1), to identify polymorphism rs 4880 (alleles C and T) in the SOD2 gene;

- a group of probes, the sequences of which are presented by SEQ ID NO 15-18 (Table 1), to identify polymorphism rs 1042522 (alleles C and G) in the TP53 gene;

- a group of probes, the sequences of which are represented by SEQ ID NOS 19-26 (Table 1), for identification of polymorphisms rs 11615 (alleles C and T), rs 3212986 (alleles G and T) in reHeERCC1;

- a group of probes, the sequences of which are presented by SEQ ID NO 27-30 (Table 1), for identification of polymorphism rs 2228001 (alleles A and C) in the CSR gene;

- a group of probes, the sequences of which are presented by SEQ ID NO 31-32 (Table 1), to identify polymorphism rs 4444903 (alleles A and G) in the EGF gene;

- a group of probes, the sequences of which are represented by SEQ ID NO 33-36 (Table 1), to identify polymorphism rs 8175347 (alleles TA5, TA6, TA7 and TA8) in the UGT1A1 gene;

- a group of probes, the sequences of which are presented by SEQ ID NO 37-46 (Table 1), for identification of polymorphisms rs 3918290 (alleles A and G), rs 2297595 (alleles C and T), rs 67376798 (alleles A and T) in the DPYD gene ;

- a group of probes, the sequences of which are presented by SEQ ID NO 47-50 (Table 1), to identify polymorphism rs 25487 (alleles A and G) in the XRCC1 gene;

- a group of probes, the sequences of which are presented by SEQ ID NO 51-52 (Table 1), to identify polymorphism rs 3892097 (alleles A and G) in the CYP2D6 gene;

- a group of probes, the sequences of which are presented by SEQ ID NO 61-66 (Table 1), for identification of polymorphisms rs 1142345 (alleles A and G), rs 1800462 (alleles G and C), rs 1800584 (alleles A and G), rs 1800460 (G and A alleles) in the TPMT gene;

b) carrying out a polymerase chain reaction for amplification of target gene fragments using a set of primers and the inclusion of a fluorescent label during the reaction to obtain predominantly single-stranded fluorescently-labeled products;

c) hybridization of the PCR products obtained in stage (b) on the biochip obtained in stage (a)

d) recording and interpreting the results of hybridization on a biochip, carried out at stage (c), by comparing the intensity of fluorescence of signals within each of the groups of cells in which perfect and imperfect hybridization duplexes were formed.

For analysis of the proposed method uses DNA isolated from human blood. PCR with a set of primers (Table 2) at stage (b), which results in the production of single-stranded target fragments of the analyzed genes and their fluorescence labeling by incorporating a fluorescent dye into the reaction product.

Results are recorded at stage (d) using a fluorescence analyzer equipped with specialized software, which allows software to automatically process signal intensities with subsequent interpretation of the results.

The invention provides a biochip used in a method for identifying genetic polymorphisms that affect the metabolism of anticancer drugs, and which is a substrate with gel elements, with a set of immobilized probes.

The invention provides a set of oligonucleotide probes for the preparation of a biochip used in a method for identifying genetic polymorphisms affecting the metabolism of antitumor drugs, the probes having the sequences of SEQ ID NOs 1-66 (Table 1).

The invention proposes a set of oligonucleotide primers for PCR, and the primers have the sequence of SEQ ID NO 67-108 (table 2).

Further, the invention will be disclosed in more detail with reference to the figures and examples, which are given solely to illustrate and explain the essence of the claimed invention, but which are not intended to limit the scope of claims.

Brief description of tables and figures Table 1. Characterization of oligonucleotide probes immobilized in biochip cells to identify genetic polymorphisms that affect the metabolism of antitumor drugs.

Table 2. The list of oligonucleotide primers used in the method for the identification of genetic polymorphisms that affect the metabolism of antitumor drugs.

FIG. 1. presents a layout of oligonucleotide probes on a biochip to identify genetic polymorphisms that affect the metabolism of antitumor drugs.

Position 0 - elements that do not contain immobilized probes (background cells); position M - elements containing a fluorescent marker;

positions 1 to 66 are elements containing immobilized oligonucleotide probes (probe sequences are shown in Table 1).

FIG. 2. presents a fluorescence image (hybridization picture) of the biochip and a program report after analysis of DNA sample No. 1.

FIG. 3. presents a fluorescence image (hybridization picture) of the biochip and the program report after analysis of DNA sample No. 2.

FIG. 4. presents a fluorescence image (hybridization picture) of the biochip and the program report after analysis of DNA sample No. 3.

FIG. 5. presents a fluorescence image (hybridization picture) of the biochip and the program report after analysis of DNA sample No. 4.

The implementation of the invention

The aim of the present invention is to provide a method for multi-parameter rapid analysis of genetic polymorphisms in genes whose products are involved in the metabolism of drugs used for chemotherapy of cancer.

Identification of genetic polymorphisms affecting the metabolism of anticancer drugs includes:

- identification of polymorphism rs 1695 (alleles A and G) in the GSTP1 gene;

- identification of polymorphism rs 1045642 (T and C alleles) in the ABCB1 gene;

- identification of polymorphism rs 1801133 (T and C alleles) in the MTHFR gene;

- identification of polymorphism rs 396991 (alleles G and T) in the FCGR3A gene;

- identification of polymorphism rs 4880 (alleles C and T) in the SOD2 gene;

- identification of polymorphism rs 1042522 (alleles C and G) in the TP53 gene;

- identification of polymorphisms rs 11615 (alleles C and T), rs 3212986 (alleles G and T) in the ERCC1 gene;

- identification of polymorphism rs 2228001 (alleles A and C) in the CSR gene;

- identification of polymorphism rs 4444903 (alleles A and G) in the EGF gene;

- identification of polymorphism rs 8175347 (alleles TA5, TA6, TA7 and TA8) in the UGT1A1 gene;

- identification of polymorphisms rs 3918290 (alleles A and G), rs 2297595 (alleles C and T), rs 67376798 (alleles A and T) in the DPYD gene;

- identification of polymorphism rs 25487 (alleles A and G) in the XRCC1 gene;

- identification of polymorphism rs 3892097 (alleles A and G) in the CYP2D6 gene;

- identification of polymorphisms rs 1142345 (alleles A and G), rs 1800462 (alleles G and C), rs 1800584 (alleles A and G), rs 1800460 (alleles G and A) in the TPMT gene;

Simultaneous multi-parameter DNA analysis is achieved using biological microarrays - arrays of elements containing oligonucleotide probes. The claimed method consists in providing a biochip with appropriate immobilized specific oligonucleotide probes, PCR using oligonucleotide primers, complementary to fragments of target gene regions, to obtain predominantly single-stranded fluorescently-labeled DNA fragments, hybridization of fluorescence-labeled products with biological interpretation and hybridization.

The biological microchip used in the proposed method is an array of three-dimensional hydrogel elements located on a solid substrate and containing covalently immobilized oligonucleotide probes. The elements of the array are microdroplets of a given volume, as a rule, with a diameter of 80 to 300 microns, the distance between the drops is, as a rule, 150-500 microns. Microchips are made by copolymerization immobilization according to the technology developed at the IMB RAS (patent US 7,846,656 B2, publication "Composition for polymerising immobilization of biological molecules and method for producing said composition", Rubina AY, Pan'kov SV, Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production, Anal. Biochem. 2004, v. 325, p. 92-106).

In the manufacture of a biochip, a polymerization mixture containing gelling monomers and oligonucleotide probes to be immobilized is applied to the substrate in the form of an array of microdrops using an automatic microdoser (robot). Gel polymerization with simultaneous covalent immobilization of probes occurs under the influence of UV radiation. The manufacture of a biochip based on hydrogels for the identification and analysis of influenza virus RNA is described in Example 1.

In FIG. 1 is a diagram of a biochip, the hydrogel elements of which contain immobilized oligonucleotide probes with the sequences SEQ ID NO 1-66 (positions 1-66). In addition, the biochip contains elements that do not contain immobilized compounds for measuring background signals (position 0), as well as elements containing a fluorescent marker (position M), for the correct positioning of the biochip when receiving a fluorescent image and image capture by the biochip analyzer software . When a fluorescently-labeled product is hybridized with oligonucleotide probes immobilized on a chip, a stable complex is formed with the most complementary probe, which leads to the appearance of a significant fluorescent signal in the corresponding biochip element. A comparative analysis of fluorescent signals allows us to conclude that the corresponding polymorphism is characteristic (homozygous for the wild / mutant allele; heterozygous).

For the analysis of this method, DNA isolated from blood obtained from humans is used. DNA isolation is carried out by any of the methods known in the art or using commercially available reagent kits and automated robotic stations, for example, Proba-GS-GENETICS reagent kit, DNA-Technology LLC, Russia, QIAamp DNA Blood Mini Kit, Qiagen, Germany, PureLink Genomic DNA Mini Kit, Thermo Scientific, USA.

At the next stage of the analysis of this method, the obtained DNA is used as a template for PCR using oligonucleotide primers complementary to the corresponding fragments of the target gene regions, a mixture of four deoxynucleoside triphosphates, fluorescently-labeled deoxynucleoside triphosphate, to obtain amplification products in the form of predominantly single-stranded fluorescence DNA A mixture of deoxynucleoside triphosphates contains dATP, dGTP, dTCP and dTTP; instead of dTTP, you can use dUTP or a mixture of dTTP with dUTP in any ratio. Any of these deoxynucleoside triphosphates may carry the fluorescent label.

As the fluorescent label, any fluorescent dye can be used that can be incorporated into the deoxynucleoside triphosphate molecule so as not to interfere with PCR and subsequent hybridization. For example, a fluorescent dye may be attached to the 5'-end of the aminoallyl derivative of dUTP. Examples of dyes include dyes of fluorescein (TAMRA, ROX, JOE), rhodamine (Texas Red), polymethine (Cy3, Cy5, Cy5.5, Cy7) series, but are not limited to.

After PCR, the products obtained are hybridized on a biochip containing immobilized oligonucleotide probes complementary to the analyzed sequences. Hybridization is carried out in a solution containing a buffer component to maintain pH, a salt to create ionic strength and a chaotropic (destabilizing hydrogen bond) agent in a sealed hybridization chamber. As a chaotropic agent, for example, guanidine thiocyanate, urea, formamide and the like can be used. The discriminating oligonucleotides of the present invention immobilized on a biochip have melting points in the range of 42 to 44 ° C, which allows hybridization at 37 ° C in the presence of a chaotropic agent.

DNA fragments form perfect hybridization duplexes only with the corresponding, fully complementary oligonucleotides. Discrimination of perfect and imperfect duplexes is performed by comparing the fluorescence intensities of the microchip cells in which the duplexes are formed. The signal intensity in cells in which perfect hybridization duplexes were formed is higher than in cells where imperfect duplexes were formed. Registration and interpretation of hybridization results on a biochip is carried out by comparing the fluorescence intensity of signals within each of the groups of cells in which perfect and imperfect hybridization duplexes are formed.

Registration of hybridization results on microchips can be carried out using any fluorescence analyzer devices equipped with specialized software that allows using software-based automatic processing of signal intensities with subsequent interpretation of the results, for example, using commercial GenePix 4000 V scanning devices (Axon Instruments, USA), 'GenePix Pro', 'Acuity' (Axon Instruments, USA), portable biochip analyzer: A universal hardware-software complex (UAPK) for biological analysis microchips (BIOCHIP-IMB LLC, Russia).

The analysis: the identification of genetic polymorphisms that affect the metabolism of antitumor drugs using biological microarrays, according to this method is described in Example 2. The results of the analysis are presented in FIG. 3-6.

This invention also provides a biochip for identifying genetic polymorphisms that affect the metabolism of antitumor drugs, which is a substrate with hydrogel elements, in each of which a set of specific oligonucleotide probes is immobilized. An example of a biochip is shown in FIG. 1 and 2, examples of analysis results using it are presented in FIG. 3-6.

The nucleotide sequences (SEQ ID NO 1-66) of the probes for immobilization in the hydrogel elements of the microchip of the invention are presented in Table 1.

The invention is further illustrated by examples that are intended to provide a better understanding of the invention, and which should not be construed as limiting the invention.

Example 1. The manufacture of a biochip based on hydrogels to identify polymorphisms that affect the metabolism of antitumor drugs. Oligonucleotide probes with sequences of SEQ ID NOS 1-66 were synthesized using a 394 DNA / RNA synthesizer (Applied Biosystems, USA).

Oligonucleotides for immobilization in the hydrogel cells of the biochip contained a spacer with the free amino group 3'-Amino-Modifier C7 CPG 500 (Glen Research, USA). The production of biochips was carried out in accordance with the procedure described previously (Rubina AY, Pan'kov SV et al. Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production. Anal. Biochem., 2004, v. 325, p. 92-106).

Polymerization mixtures containing a mixture of gelling monomers (methacrylamide derivatives, methylene bis acrylamide, stabilizers, etc.) and immobilized compounds (oligonucleotides or IMD 504 fluorescent dye) were placed in cells of a 384-well plate. Using a QArray robot (Genetix, UK), mixtures from the wells of the tablet were transferred as microdrops onto the surface of plastic substrates. Gel polymerization with simultaneous covalent immobilization of oligonucleotides in the gel structure occurred under the influence of UV radiation (UV lamp Sylvania GTE F15T8350 BL, UK), 40 min, 20 ° С, in a stream of nitrogen. After polymerization, the substrates with arrays of hydrogel cells (microdrops) were washed with 0.1 M phosphate buffer, water and dried in a dustless atmosphere.

The resulting biochips were a set of hemispherical cells with a diameter of 100 μm, the distance between cells (period) - 300 μm. The biochip structure is shown in FIG. 12.

The microchip contains 76 hydrogel elements. Elements 1-66 (Fig. 1) contain immobilized oligonucleotide probes with the sequences SEQ ID NO 1-66; indicated in Table 1;

cells 1-4 contain oligonucleotide probes (SEQ ID NO 1-4) corresponding to a fragment of the target sequence of the GSTP1 gene for determining allelic variants of polymorphism rs 1695 (alleles A and G). Cells 1, 4 contain probes specific for the allele A, cells 2, 3 - for the allele G;

cells 5, 6 contain oligonucleotide probes (SEQ ID NO 5, 6) corresponding to a fragment of the target ABCB1 gene sequence for determining allelic variants of rs 1045642 polymorphism (T and C alleles). Cell 5 contains probes specific to the T allele, cell 6 to the C allele;

cells 7, 8 contain oligonucleotide probes (SEQ ID NO 7, 8) corresponding to a fragment of the target sequence of the MTHFR gene for determining allelic variants of rs 1801133 polymorphism (T and C alleles). Cell 7 contains probes specific for the T allele, cell 8 - for the C allele;

cells 9, 10 contain oligonucleotide probes (SEQ ID NO 9, 10) corresponding to a fragment of the target sequence of the FCGR3A gene for determining allelic variants of rs 396991 polymorphism (G and T alleles). Cell 9 contains probes specific for the G allele, cell 10 for T alleles;

cells 11-14 contain oligonucleotide probes (SEQ ID NO 11-14) corresponding to a fragment of the target sequence of the SOD2 gene for determining allelic variants of the rs 4880 polymorphism (C and T alleles). Cells 11, 13 contain probes specific for the C allele, cells 12, 14 - for the T allele;

cells 15-18 contain oligonucleotide probes (SEQ ID NO 15-18) corresponding to a fragment of the target sequence of the TP53 gene for determining allelic variants of rs 1042522 polymorphism (C and G alleles). Cells 15, 17 contain probes specific for the C allele, cells 16, 18 - for the G allele;

cells 19-26 contain oligonucleotide probes (SEQ ID NO 19-26) corresponding to a fragment of the target sequence of the ERCC1 gene for determining allelic variants of polymorphisms rs 11615 (alleles C and T), rs 3212986 (alleles G and T). Cells 23, 24 contain probes specific to the C allele, cells 25, 26 to the T allele (polymorphism rs 11615). Cells 19, 21 contain probes specific for the G allele, cells 20, 22 - for the T allele (polymorphism rs 3212986);

cells 27-30 contain oligonucleotide probes (SEQ ID NO 27-30) corresponding to a fragment of the target sequence of the XPC gene for determining allelic variants of polymorphism rs 2228001 (alleles A and C). Cells 27, 29 contain probes specific for the allele A, cells 28, 30 for the allele C;

cells 31, 32 contain oligonucleotide probes (SEQ ID NO 31, 32) corresponding to a fragment of the target EGF gene sequence for determining allelic variants of polymorphism rs 4444903 (alleles A and G). Cell 31 contains probes specific for allele A, cell 32 for G allele;

cells 33-36 contain LNA-modified oligonucleotide probes (SEQ ID NO 33-36) corresponding to a fragment of the target sequence of the UGT1A1 gene for determining allelic variants of the rs 8175347 polymorphism (alleles TA5, TA6, TA7 and TA8); Cell 33 contains probes specific for the TA5 allele, cell 34 for TA6, cell 35 for TA7, cell 36 for TA8;

cells 37-46 contain oligonucleotide probes (SEQ ID NO 37-46) corresponding to a fragment of the target sequence of the DPYD gene for determining allelic variants of polymorphisms rs 3918290 (alleles A and G), rs 2297595 (alleles C and T), rs 67376798 (alleles A and T). Cell 37 contains probes specific for the A allele, cell 38 contains G-specific alleles (polymorphism rs 3918290). Cells 43, 45 contain probes specific for the C allele, cells 44, 46 for the T allele (polymorphism rs 2297595). Cells 39, 51 contain probes specific for the allele A, cells 40, 42 for the T allele (polymorphism rs 67376798);

cells 47-50 contain oligonucleotide probes (SEQ ID NO 47-50) corresponding to a fragment of the target sequence of the XRCC1 gene to determine allelic variants of polymorphism rs 25487 (alleles A and G). Cells 47, 49 contain probes specific for the allele A, cells 48, 50 for the allele G;

cells 51, 52 contain oligonucleotide probes (SEQ ID NO 51, 52) corresponding to a fragment of the target sequence of the CYP2D6 gene for determining allelic variants of polymorphism rs 3892097 (alleles A and G). Cell 51 contains probes specific for the allele A, cell 52 for the allele G;

cells 53-66 contain oligonucleotide probes (SEQ ID NO 53-66) corresponding to a fragment of the target sequence of the TPMT gene for determining allelic variants of polymorphisms rs 1142345 (alleles A and G), rs 1800462 (alleles G and C), rs 1800584 (alleles A and G), rs 1800460 (alleles G and A). Cells 53, 55 contain probes specific for the allele A, cells 40, 42 for the allele G (polymorphism rs 1142345). Cells 57, 59 contain probes specific for the G allele, cells 58, 60 - for the C allele (polymorphism rs 1800462). Cell 61 contains probes specific for the A allele, cell 62 - for the G allele (polymorphism rs 1800584). Cells 63, 65 contain probes specific to the G allele, cells 64, 66 to the A allele (polymorphism rs 1800460).

The composition of the chip also includes marker points containing an immobilized fluorescent dye IMD 504 (BIOCHIP LLC, Moscow) (elements 1 in Fig. 1) for the correct positioning of the biochip, performed by a computer program when analyzing the fluorescence image after hybridization, and the cell, not containing immobilized compounds (elements 0 in Fig. 1), necessary for calculating the background value of the fluorescence intensity.

Example 2. The analysis procedure: the identification of genetic polymorphisms that affect the metabolism of anticancer drugs using a biological microchip.

The analysis included the stages of: preparing DNA samples; PCR amplification to obtain single-stranded fluorescently-labeled DNA fragments; hybridization of amplified fluorescently-labeled products on biochips; registration and interpretation of hybridization results,

a) Preparation of DNA samples

Whole peripheral blood was taken in 10 ml Vacuette type plastic vacuum tubes with ethylene diamine tetraacetate disodium salt (EDTA) added as an anticoagulant at a final concentration of 2.0 mg / ml. To mix blood with an anticoagulant after taking the material, the tube was inverted 2-3 times.

DNA samples were isolated from peripheral blood using the Proba-GS-GENETICS kit DNA-Technology LLC, Russia.

1) A mixture of a lysing solution with a sorbent was prepared by mixing in a separate tube:

- 150 * (N + 1) μl of lysis solution;

- 20 * (N + 1) μl of a previously resuspended sorbent;

where N + 1 - the number of analyzed samples taking into account "K-" with a margin of 1 sample;

2) 170 μl of the resulting mixture were added to 1.5 ml tubes;

3) 100 μl of peripheral blood was added to each of the tubes with the mixture (except for the K- tube);

4) in a test tube "K-" was added 100 μl of sterile saline;

5) Tightly closed tubes were shaken on a vortex for 3-5 s;

6) the tubes were incubated in a thermostat for 10 min at + 50 ° C;

7) centrifuged tubes at 13,000 rpm for 1 min;

8) without touching the pellet, the supernatant was completely removed (with a separate tip from each tube);

9) 400 μl of Wash Solution No. 1 was added to the pellet and the vortex tubes were shaken for 3-5 s;

10) centrifuged tubes at 13,000 rpm for 1 min;

11) without touching the pellet, the supernatant was completely removed (with a separate tip from each tube);

12) 200 μl of Wash Solution No. 2 was added to the pellet and the tubes were vortexed for 3-5 s, the steps were repeated. 10, 11;

13) repeated item 12 once;

14) by opening the lids of the tubes, the precipitate was dried at + 50 ° C for 5 min;

15) 300 μl of the elution solution were added to the pellet and the vortex tubes were shaken for 5–10 s;

16) tubes were incubated at + 50 ° C for 5 min;

17) tubes were centrifuged at 13,000 rpm for 1 min. and transferred the supernatant to a new tube.

The obtained DNA samples were used as a template for PCR

b) Preparation of primers for PCR

When selecting primer sequences for PCR, nucleotide sequence databases known to those skilled in the art were used, for example, the database http://www.ncbi.nlm.nih.gov/Genbank/index.html. The specificity of the primers was checked, for example, using software using a search in the nucleotide sequence databases using the BLAST algorithm (for example, www.ncbi.nlm.nih.gov/BLAST).

The primers used in this example are listed in Table 2. The primers were synthesized using a 394 DNA / RNA synthesizer (Applied Biosystems, USA). As a fluorescent label, we used dye Imd 500-dUTP No. 49, d-UTP conjugate (BIOCHIP-IMB LLC), maximum absorption at 645 nm, maximum fluorescence at 670 nm. Labeling was carried out in accordance with the manufacturer's recommendations.

c) PCR for amplification of target gene fragments

The reaction mixture was prepared for PCR, introducing the following reagents into the test tube (based on 1 reaction):

- deionized water 21.5 μl;

- PCR buffer (10 × multiple mixture containing 100 tM Tris-HCl, 500 mM KCl; 15 mM MgCl ₂ ) 3 μl;

- dNTP (an aqueous solution of deoxynucleoside triphosphates containing 2 mmol / l of DATP, dGTF, dUTP, dTTP) 3 μl;

- a mixture of primers 1 μl;

- Taq polymerase, activity 5 Units / μl 0.5 μl.

To 29 μl of the mixture was added 1 μl of DNA obtained after the DNA extraction step. For the negative control sample, deionized sterile water was added instead of the matrix. The tubes were placed in a C1000 DNA amplifier, Biorad, USA, and PCR was performed according to the following program: preliminary DNA denaturation: + 95 ° С, 1 cycle 10 min; 39 PCR cycles according to the following temperature-time regime: + 95 ° С - 30 s, + 70 ° С - 30 s, + 72 ° С - 30 s; 42 PCR cycles according to the following temperature-time regime: + 95 ° С - 30 s, + 54 ° С - 30 s, + 72 ° С - 30 s; final elongation at + 72 ° C - 5 min.

d) Hybridization of fluorescently-labeled products on biochips

To 27 μl of the reaction mixture obtained after PCR, a concentrated solution of hybridization buffer was added so that the final concentration of the components was:

- guanidine thiocyanate - 1M,

- HEPES - 50 mm (pH 7.5),

- EDTA - 5 mm.

30 μl of the resulting mixture was placed in the hybridization chamber of the microchip through an opening in the chamber. Biochips were placed in a thermostat at + 37 ° C and incubated for 5 hours. After hybridization, the hybridization chambers were removed and the biochips were washed three times with distilled water heated to + 37 ° C for 30 s and dried.

e) Registration and interpretation of hybridization results

Hybridization results were recorded using a universal hardware-software complex (UAPK) for image analysis of diagnostic microarrays using the specialized ImageWare software (BIOCHIP-IMB LLC).

Fluorescence images of biochips after hybridization are shown in FIG. 3-6.

Interpretation of hybridization results on a biochip is carried out according to an algorithm implemented in a software module by comparing the fluorescence intensity of signals within each of the groups of cells in which perfect and imperfect hybridization duplexes are formed.

Example 3. Identification of genetic polymorphisms that affect the metabolism of antitumor drugs. For the analysis used biological microarrays obtained as described in Example 1. The analysis procedure is described in Example 2. Fluorescence images (hybridization patterns) and the information given in the program reports are presented in Figures 3-6.

The correctness of the determination of allelic variants of polymorphisms in the analyzed genes by the proposed method in all cases was checked by an independent method of sequencing nucleotide sequences of target gene fragments. Sequencing was performed on a 3130xL Applied Biosystems automated sequencer (USA) using the BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit commercial sequencing kit. As sequencing primers, the corresponding primers used for PCR were used.

The sign "*" after the nucleotide means that the nucleotide is LNA-modified.

Claims (23)

1. A method for identifying genetic polymorphisms that affect the metabolism of antitumor drugs, including: a) providing a biochip, which is a substrate with gel elements, in each of which a specific oligonucleotide probe is immobilized, belonging to one of the following groups: - a group of probes, the sequences of which are represented by SEQ ID NO 1-4, to identify the polymorphism rs1695 (alleles A and G) in the GSTP1 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 5, 6, for identifying the polymorphism rs1045642 (T and C alleles) in the ABCB1 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 7, 8, for identifying the polymorphism rs1801133 (T and C alleles) in the MTHFR gene; - a group of probes, the sequences of which are represented by SEQ ID NO 9, 10, to identify the polymorphism rs396991 (alleles G and T) in the FCGR3A gene; - a group of probes, the sequences of which are represented by SEQ ID NO 11-14, to identify the polymorphism rs4880 (C and T alleles) in the SOD2 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 15-18, to identify the polymorphism rs1042522 (alleles C and G) in the TP53 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 19-26, for the identification of polymorphisms rs11615 (alleles C and T), rs3212986 (alleles G and T) in the ERCC1 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 27-30, for the identification of polymorphism rs2228001 (alleles A and C) in the CSR gene; - a group of probes, the sequences of which are represented by SEQ ID NO 31-32, to identify polymorphism rs4444903 (alleles A and G) in the EGF gene; - a group of probes, the sequences of which are represented by SEQ ID NO 33-36, to identify the polymorphism rs8175347 (alleles TA5, TA6, TA7 and TA8) in the UGT1A1 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 37-46, to identify polymorphisms rs3918290 (alleles A and G), rs2297595 (alleles C and T), rs67376798 (alleles A and T) in the DP YD gene; - a group of probes, the sequences of which are represented by SEQ ID NO 47-50, to identify polymorphism rs25487 (alleles A and G) in the XRCC1 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 51-52, to identify polymorphism rs3892097 (alleles A and G) in the CYP2D6 gene; - a group of probes, the sequences of which are represented by SEQ ID NO 61-66, for the identification of polymorphisms rs1142345 (alleles A and G), rs1800462 (alleles G and C), rs1800584 (alleles A and G), rs1800460 (alleles G and A) in the gene TRMT; b) a set of oligonucleotide primers complementary to the target fragments of the GSTP1, ABCB1, MTHFR, FCGR3A, SOD2, TP53, ERCC1, ХРС, EGF, UGT1A1, DPYD, XRCC1, CYP2D6, TPMT gene fragments, which provide predominantly single-chain products DNA c) hybridization of the amplified fluorescently-labeled products obtained in stage (b), on the biochip obtained in stage (a); d) recording and interpreting the results of hybridization on a biochip carried out in stage (c) by comparing the fluorescence intensity of signals within each of the groups of cells in which perfect and imperfect hybridization duplexes were formed. 2. The method according to p. 1, characterized in that the analysis uses DNA isolated from human peripheral blood. 3. The method according to p. 1, characterized in that the registration of the results of stage (d) is carried out using a fluorescence analyzer equipped with specialized software for this method, which allows the use of automatic processing of signal intensities with subsequent interpretation of the results. 4. The biochip used in the method for identifying genetic polymorphisms that affect the metabolism of antitumor drugs, which is a substrate with gel elements, each of which has a specific probe immobilized, moreover, the probe sequences are represented by SEQ ID NOs 1-66. 5. The set of oligonucleotide probes used to obtain the biochip, in a method for identifying genetic polymorphisms that affect the metabolism of antitumor drugs, and the probes have the sequences SEQ ID NO 1-66.

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